Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics

ABSTRACT: Enzymes that bind and process ubiquitin, a small 76 amino acid protein, have been recognized as pharmacological targets in oncology, immunological disorders and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition of the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation and more potent/selective ubiquitin active-site probes with propargyl based electrophiles to profile 74 DUBs including distinguishable isoforms for five DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents, pan-DUB inhibitors ubiquitin combined with probe labelling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry, demonstrating that USP13, 39 and USP40 are non-reactive to probe, reflecting no, low or restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small molecule DUB selectivity profiling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE134954 | GEO | 2019/08/31

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Comprehensive landscape of active deubiquitinating enzymes profiled by advanced chemoproteomics

Project description:Enzymes that bind and process ubiquitin, a small 76 amino acid protein, have been recognized as pharmacological targets in oncology, immunological disorders and neurodegeneration. Mass spectrometry technology has now reached the capacity to cover the proteome with enough depth to interrogate entire biochemical pathways including those that contain DUBs and E3 ligase substrates. We have recently characterized the breast cancer cell (MCF7) deep proteome by detecting and quantifying ~10,000 proteins, and within this data set, we can detect endogenous expression of 65 deubiquitylating enzymes (DUBs), whereas matching transcriptomics detected 78 DUB mRNAs. Since enzyme activity provides another meaningful layer of information in addition of the expression levels, we have combined advanced mass spectrometry technology, pre-fractionation and more potent/selective ubiquitin active-site probes with propargylic based electrophiles to profile 74 DUBs including distinguishable isoforms for five DUBs in MCF7 crude extract material. Competition experiments with cysteine alkylating agents, pan-DUB inhibitors combined with probe labelling revealed the proportion of active cellular DUBs directly engaged with probes by label-free quantitative (LFQ) mass spectrometry. This demonstrated that USP13, 39 and 40 are non-reactive to probe, indicating restricted enzymatic activity under these cellular conditions. Our extended chemoproteomics workflow increases depth of covering the active DUBome, including isoform-specific resolution, and provides the framework for more comprehensive cell-based small molecule DUB selectivity profiling.

2019-11-12 | PXD014391 | Pride

USP7: combing tools towards selectivity

Project description:Active-site probes based on the ubiquitin scaffold have been successfully applied as tools to determine the levels of active deubiquitinating enzymes (DUBs). Here, we show the development of a Ub-based reagent selective for the DUB USP7. This concept can be applied for the generation of other new selective reagents.

2019-03-13 | PXD012535 | Pride

Proteomics-based identification of DUBsubstrates using selective inhibitors

Project description:Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here we demonstrate that recently developed potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this methodology to USP7, a DUB widely investigated as a therapeutic target and identified novel substrates. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general methodology widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.

2021-04-06 | PXD020881 | Pride

Proteomics-based identification of DUBsubstrates using selective inhibitors

2021-01-18 | PXD020865 | Pride

Proteomics-based identification of DUBsubstrates using selective inhibitors

2021-01-18 | PXD020873 | Pride

Proteomics-based identification of DUBsubstrates using selective inhibitors

2021-01-18 | PXD020882 | Pride

Transcriptomics following CRISPR KO of individual DUBs or inhibition of DUBs with small molecule inhibitors

Project description:CRISPR KO RNA-seq data: To generate knockouts, we used a commercially available arrayed CRISPR-Cas9 library targeting 81 out of ~100 DUBs and 13 additional proteins in the ubiquitin-proteasome system, including ubiquitin-like proteins; the library was constructed with four, pooled, guides per target. mRNA profiling was performed 96 hours after guide RNA transfection using a high-throughput, low-cost RNA-sequencing method (3’ Digital Gene Expression or 3’DGE-seq) in the MDAMB231 breast cancer cell line. Inhibitor RNA-seq data: For the small molecule inhibitor signatures, MDAMB231 or MCF7 or MCF7 TP53 shRNA breast cancer cells were treated with a panel of DUB inhibitors for 24 hours, then 3'DGE-seq was used to perform the mRNA profiling.

2021-11-03 | GSE187008 | GEO

Electrophile scanning by chemical prote-omics reveals a potent pan-active DUB probe for investigation of deubiquitinase activity in live cells

Project description:Deubiquitinases (DUBs) are proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, which can lead to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been im-plicated in many disease states, including cancer, neurodegeneration and inflammation, making them potentially attractive targets for therapeutic intervention. The >100 known DUB enzymes have been classified primarily by their conserved active sites, but we are still building our understanding of their substrate profiles, localization and regulation of DUB activity in di-verse contexts. Ubiquitin-derived covalent activity-based probes (ABPs) are the premier tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which account for local DUB concentration, protein interactions, complexes, and organelle compartmentalization in intact cells or organisms. Here, through comprehensive warhead profiling we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12), a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cell systems. Through chemical prote-omics and targeted assays we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs in live cells across a range of non-toxic concentrations, and in diverse cell lines and disease models, including induction of MYC deregulation in B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the con-text of disease-relevant phenotypes.

2024-01-03 | PXD035417 | Pride

Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [CRISPR]

Project description:The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1 isoform, and while USP43 does not alter HIF-1 stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia-dependent manner to increase the nuclear pool of HIF-1, independently of DUB activity. Together, our results unveil the DUB landscape in HIF signalling, and highlight the multifunctionality of DUBs, illustrating that they can provide important signalling roles outside of their catalytic activity.

2024-05-17 | GSE252651 | GEO

Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [RNA-seq]

2024-05-17 | GSE252654 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data